Quantitation of N^ϵ-(dichloroacetyl)-l-lysine in proteins after perchloroethene exposure by gas chromatography–mass spectrometry using chemical ionization and negative ion detection following immunoaffinity chromatography

Abstract

An antibody specific to N^ϵ-(dichloroacetyl)-l-lysine (DCA-Lys) was immobilized to immunoaffinity columns for the use in selective enrichment of dichloroacetylated proteins. These result from the reaction with dichlorothioketene the β-lyase cleavage product of the perchloroethene metabolite S-(trichlorovinyl)-l-cysteine. Dichloroacetylated proteins from rat kidney mitochondria, rat plasma and human blood plasma were isolated after exposure to 40 ppm tetrachloroethene (PER) for 6 h. After acid hydrolysis of the protein fraction, DCA-Lys was derivatized with 1,3-dichloro-1,1,3,3-tetrafluoroacetone using N^ϵ-(trifluoroacetyl)-l-lysine as internal standard. Recovery of dichloroacetylated reference proteins from immunoaffinity columns was about 73%. Samples were analyzed by GC–MS with chemical ionization and negative ion (NCI) detection showing DCA-Lys in proteins with 2.26 (±0.02) pmol/mg protein in male rat kidney mitochondria and 1.92 (±0.05) pmol/mg total mitochondrial protein in female rats. In rat plasma 0.47 (±0.006) pmol DCA-Lys/mg protein in male and 0.34 (±0.02) in female animals were found. DCA-Lys could not be detected in blood plasma of human volunteers exposed to PER with a detection limit of 20 fmol for the DCA-Lys derivative 2,2-bis(chlorodifluoromethyl)-4-(1-dichloroacetamido)-butyl-1,3-oxazolidine-5-one. Immunoaffinity chromatography with specific antibodies provides a powerful tool for the enrichment of minor quantities of dichloroacetyled proteins in biological samples for GC–NCI-MS analysis of the modified amino acid lysine having broad utility in the biomonitoring of PER exposure.

Introduction

Perchloroethene (tetrachloroethene, PER) (Fig. 1, $\text{1}$) is extensively used in industry as a metal degreasing solvent and as dry cleaning agent. Due to its volatility and resistance to degradation, it is a widely distributed environmental air pollutant and groundwater contaminant [1].

Long-term exposure of rodents to PER has been shown to increase the incidence of liver tumors in male mice and to result in a small but significant increase in the incidence of renal tumors in male rats [2]. The chronic toxicity of PER is most likely mediated by bioactivation reactions. PER is known to be metabolized by both cytochrome P450 and glutathione-dependent biotransformation pathways leading to the generation of reactive metabolites which may covalently bind to cellular macromolecules (Fig. 1). Cytochrome P450 oxidation of PER results in formation of trichloroacetyl chloride which reacts with amino groups in macromolecules [3], [4]. In addition, glutathione conjugation of PER followed by cysteine conjugate β-lyase-mediated activation of S-(trichlorovinyl)-l-cysteine (Fig. 1, $\text{2}$) to the ultimate metabolite dichlorothioketene $\text{3}$ likely responsible for the nephrotoxicity and possible renal tumorigenicity of PER [5], [6], [7], [8]. Both N^ϵ-(dichloroacetyl)-l-lysine (Fig. 1, $\text{4}$) and N^ϵ-(trichloroacetyl)-l-lysine have been identified as modified amino acids in proteins by GC–MS in the liver and kidneys of rats treated with PER [4].

Up to now, the main difficulty in the identification and detection of protein modifications linked to chemical or metabolite interactions in tissues are the large amounts of unmodified proteins and the enrichment of sufficient quantities for instrumental analysis. Separation of modified proteins from native proteins is the most challenging problem. In the recent years several immunochemical methods have become available to detect and quantify trace levels of protein modifications after chemical exposure. Antibodies to reactive metabolite-induced protein alterations have been developed to selectively enrich the covalent adducts by immunoaffinity chromatography [9], [10]. In combination with radioimmunoassays a more sensitive detection as compared to classical methods, i.e. liquid chromatographic separation using fluorescence detection or GC–MS, was achieved. The detection of minor quantities of protein adducts by such methods using monoclonal antibodies against even small protein modifications after reaction with nitrogen oxides were reported [11]. We recently developed monospecific antibodies to N^ϵ-(dichloroacetyl)-l-lysine and to N^ϵ-(trichloroacetyl)-l-lysine in proteins [12]. The anti-N^ϵ-(dichloroacetyl)-l-lysine antibody was used for immunoaffinity enrichment of N^ϵ-(dichloroacetyl)-l-lysine containing proteins for quantitation of N^ϵ-(dichloroacetyl)-l-lysine by GC–negative ion chemical ionization (NCI) MS after electrophore derivatization in the kidney and in blood of rats and in blood of humans exposed to PER. The obtained data comparatively quantify biologically effective doses of PER and provide further evidence for a much lower extent of β-lyase-dependent bioactivation of PER-metabolites in humans as compared to rats.